Identification of critical active‐site residues in angiotensin‐converting enzyme‐2 (ACE2) by site‐directed mutagenesis

Abstract

Angiotensin-converting enzyme-2 (ACE2) is a membrane protein with its active site exposed to the extracellular surface of endothelial cells, the renal tubular epithelium and also the epithelia of the lung and the small intestine [1][2][3]. Here ACE2 is poised to metabolize circulating peptides which may include angiotensin II, a potent vasoconstrictor and the product of angiotensin I cleavage by angiotensin-converting enzyme (ACE; EC 3.4.15.1) [1,4]. Indeed, ACE2 has been implicated in the regulation of heart and renal function where it is proposed to control the concentrations of angiotensin II relative to its hypotensive metabolite, angiotensin-(1-7) [5][6][7][8][9][10][11][12][13]. Most recently, ACE2 has been identified as a functional receptor for the coronavirus which causes the severe acute respiratory syndrome (SARS) [14]. For recent reviews, see [15,16].ACE2 shares a number of characteristics with ACE, both being zinc-containing enzymes which are sensitive to anion activation [4,17,18]. However, unlike ACE, ACE2 functions as a carboxypeptidase and is not susceptible to inhibition by the classical ACE inhibitors [1,2]. After the elucidation of the crystal structure of testicular ACE (tACE), [19] a model of the active site of ACE2 was described which demonstrated the structural determinants underlying these differences in enzyme activity [17]. Critical residue substitutions were highlighted that gave rise to the elimination of the S2¢ pocket found in ACE such that ACE2 is able to remove only a single amino acid from the C-terminus of its substrates (whereas ACE is a peptidyl dipeptidase). Shortly after this, the structure of ACE2 was solved [20] which provided further insights into this enzyme in relation to its counterpart. However, it has Angiotensin-converting enzyme-2 (ACE2) may play an important role in cardiorenal disease and it has also been implicated as a cellular receptor for the severe acute respiratory syndrome (SARS) virus. The ACE2 activesite model and its crystal structure, which was solved recently, highlighted key differences between ACE2 and its counterpart angiotensin-converting enzyme (ACE), which are responsible for their differing substrate and inhibitor sensitivities. In this study the role of ACE2 active-site residues was explored by site-directed mutagenesis. Arg273 was found to be critical for substrate binding such that its replacement causes enzyme activity to be abolished. Although both His505 and His345 are involved in catalysis, it is His345 and not His505 that acts as the hydrogen bond donor ⁄ acceptor in the formation of the tetrahedral peptide intermediate. The difference in chloride sensitivity between ACE2 and ACE was investigated, and the absence of a second chloride-binding site (CL2) in ACE2 confirmed. Thus ACE2 has only one chloride-binding site (CL1) whereas ACE has two sites. This is the first study to address the differences that exist between ACE2 and ACE at the molecular level. The results can be applied to future studies aimed at unravelling the role of ACE2, ...

A critical role for the histidine residues in the catalytic function of acyl‐CoA:cholesterol acyltransferase catalysis: Evidence for catalytic difference between ACAT1 and ACAT2

“…serine esterase, ACAT, and LCAT, possess serine, histidine, and aspartic acid residues in the active site as a catalytic triad [25][26][27]. Histidine residues are frequently found in general acid-base catalysis [28,29], reaction intermediate stabilization [30][31][32] through hydrogen bonding of their NE2 nitrogen atom. Recently, the X-ray structure has been determined of OMPLA [33], an integral membrane enzyme, where the active center is located in the outer leaflet of the membrane and contains a histidine residue as part of the catalytic triad [34].…”

β‐amino acid substitution to investigate the recognition of angiotensin II (AngII) by angiotensin converting enzyme 2 (ACE2)

“…Although substrate sequence specificity has been investigated and models for the catalytic mechanism have been proposed based on the inhibitor-bound ACE2 structure (Guy et al, 2003;Towler et al, 2004;Guy et al, 2005), information on the structural features of AngII that are involved in ACE2 recognition is lacking. Given the importance of the AngII-ACE2 interaction for regulating cardiovascular tone and the involvement of ACE2 in certain cardiovascular and liver disease, it is important to understand the structural determinants of AngII that facilitate ACE2 binding and proteolytic cleavage.…”

Angiotensin-Converting Enzyme 2 Metabolizes and Partially Inactivates Pyr-Apelin-13 and Apelin-17

“…17 We performed knowledgebased docking using our understanding from ACE-substrate bound complex structure. For this purpose, we have used the Angiotensin II (Ang II)-bound ACE complex (PDB ID: 4APH) 18 and MLN-4760 inhibitor bound ACE2 complex (PDB ID: 1R4L). 16,[18][19][20] We modeled the ACE2-Ang II, ACE2-pyr-apelin 13, and ACE2-apelin 17 complexes using these 2 above mentioned reference structures as a template.…”